Promesostoma, Graff, 1882

Diez, Yander L., Monnens, Marlies, Wuyts, Arlien, Brendonck, Luc, Reygel, Patrick, Schmidt-Rhaesa, Andreas & Artois, Tom, 2023, Taxonomy and phylogeny of Dalytyphloplanida Willems et al., 2006 (Platyhelminthes: Rhabdocoela), with the description of a new family, a new genus, and sixteen new species from Cuba and Panama, Organisms Diversity & Evolution (New York, N. Y.) 23 (4), pp. 631-681 : 673-675

Promesostoma

Currently, 37 species of Promesostoma are described (WoRMS, 2023). They all have a stylet that basically is a single-walled tube, situated in an elongated male atrium that is widened proximally. The male atrium is constricted by at least one sphincter ( Karling, 1967; Luther, 1943). Species of Promesostoma are distinguished from each other based on size and shape of the stylet, and some easily observable features of the male genital system, such as the presence of a copulatory bursa ( Karling, 1967). Unfortunately, apart from that of P. marmoratum (studied by Luther, 1943), the internal morphology is not known in detail for any of the species, as most species are only described on observations on live animals and on the detailed morphology of the stylet ( Karling, 1967). This lack of knowledge impedes the elaboration of sound hypotheses on the evolution of morphological traits within this speciose genus.

Within Promesostoma, Karling (1967) delimited some more or less homogeneous species groups based on features of the male genital system, including stylet shape (straight, curved, spiralised, branched, proximally bent or not) and position (lying completely caudal to the pharynx or next to it, or with the proximal end anterior to the pharynx and the distal end caudal to it). Two additional important taxonomic features are the presence or absence of the male copulatory bursa and of a seminal receptacle. The latter two features are easily observable in live specimens. The species groups proposed by Karling (1967) could not be retrieved in our phylogeny (see our clade ‘ cochleare ’, for example). However, our study corroborates Karling’s hypothesis about the ancestral morphological state of the male atrial organs in Promesostoma . This affirmation is supported by the fact that P. cubanum sp. n. is recovered as the sister taxon of all other species of Promesostoma in our phylogeny as this species shows the hypothetical ancestral characteristics mentioned by Karling (1967): 1, prostate vesicle provided with a short and straight stylet; 2, prostate vesicle oriented backwards and its axis is the same of that of the stylet; and 3, proximally, the stylet enlarge to a funnel with an annular thickening.

Because of the fact that their stylet is straight to slightly curved, without spines, and located caudal to the pharynx, Promesostoma cubanum sp. n. and P. cubitum sp. n. resemble P. bilineatum Pereyaslawzewa, 1892 ; P. gallicum Ax, 1956 ; P. kergroixense Ehlers & Sopott-Ehlers, 1989 ; P. norvegicum Karling, 1967 ; P. serpentistylum Ax, 1952 ; and P. tenebrosum Ax & Ehlers, 1973 . Unfortunately, except for P. cubanum sp. n., none of these species could be included in our phylogenetic analysis. Promesostoma cubanum sp. n. and P. cubitum sp. n. are unique by the fact that their prostate vesicle is located caudal to the pharynx and is connected by a short duct to the stylet, whereas the prostate vesicle is located anterior to the pharynx and is connected to the stylet by a very long duct in the other forementioned species. In this group, P. bilineatum and P. kergroixense are the only species where the stylet carries one distal wing, a feature not seen in our specimens, nor in any other species of this group. The longest stylet among these species is found in P. bilineatum and P. gallicum (240–250 µm long) ( Ax, 1952, 1956). The stylet of P. cubitum sp. n. (108–123 µm) is slightly smaller than that of P. norvegicum (130 µm), but longer than that of P. cubanum sp. n. (93–114 µm), P. kergroixense (95–100 µm), and P. tenebrosum (45–48 µm). There are no measures available in literature for the stylet in P. serpentistylum . However, it differs from that of P. cubanum sp. n. by the fact it is slender, with the same width all over its length, and has an oblique distal end (see Ax, 1952; Karling, 1967; Ehlers, 1974).

Promesostoma cubitum sp. n. is the only species of this species group that has only one seminal vesicle, whereas there is a pair of them in all other species. Furthermore, the proximal elbow-shaped projection of the stylet of P. cubitum sp. n. (Figs. 13b and 15c) is also unique.

Apart from the differences mentioned above, P. cubanum sp. n. differs from each of the other species in this group in several details. It is the only species of the group without male bursa, and it is the only one that has a muscular bulb with a proximal sperm-containing vesicle connected to the genital atrium (Fig. 13d: x). Promesostoma norvegicum has a strongly curved stylet, with the distal end irregularly club shaped and a tooth-like edge, which is not the case in P. cubanum sp. n. In P. tenebrosum the stylet has a proximal funnel surrounded by three sphincters and an oblique distal end. Moreover, it is the only species within the group that lacks eyes. The combination of these features unambiguously differentiates P. tenebrosum from P. cubanum sp. n., which has eyes, a single proximal sphincter around the funnel of the stylet, and a stylet with a rounded distal end.

Clade ‘ cochleare ’ of Promesostoma ( Fig. 22 ) encompasses two species that Karling (1967) included in two different morphological groups ( P. cochleare and P. maculosum ). In both species, the stylet is positioned caudal to the pharynx, a copulatory bursa is present, and a seminal receptacle is absent, a combination of features that also occurs in species from other clades. The same combination of features is shown by P. toryne sp. n., which has a distally split stylet. Therefore, we hypothesise that this species belongs to clade ‘ cochleare ’. A stylet with a distally split end is also found in P. balticum Luther, 1918 ; P. bipartitum Ax, 1956 ; P. furcatum Armonies, 2023 ; P. paracochlearis Ax, 1952 ; and P. wehrenbergi Armonies, 2018 . Because of this combination of characters, we conjecture that the previously mentioned species belong to clade ‘ cochleare ’. However, P. balticum has a seminal receptacle, distinguishing it from the other species mentioned above.

The species belonging to clade ‘ cochleare ’ differ from each other as to stylet length and relative position of the distal split in the stylet (see Armonies, 2018, 2023). The largest stylet occurs in P. paracochleare (210–233 μm; Ax, 1952) and the smallest in P. bipartitum (80 μm; Ax, 1956). The stylet of P. toryne sp. n. (109 μm long) is of the same length as that in P. furcatum and similar to that of P. wehrenbergi (110–112 μm). Stylet length highly varies in P. balticum (55–135 μm; Luther, 1918) and P. cochleare (120–155 μm; Karling, 1935; Ax, 1952; Luther, 1962). The fact that the stylet splits near its midpoint is characteristic for P. toryne sp. n., P. bipartitum , and P. wehrenbergi , whereas it splits more distally in P. furcatum . Furthermore, the stylet of P. toryne sp. n. is spoon-shaped distally, which, apart from this species, is only the case in P. bipartitum . However, both species can easily be distinguished from each other as P. bipartitum has paired seminal vesicles and an interposed prostate vesicle, whereas P. toryne sp. n. has a single seminal vesicle and a free prostate vesicle, the last feature being unique within the genus. The stylet of P. furcatum differs from that of P. toryne sp. n. in its proximal S-shaped end and the fact that the branched plate surrounds the main tubular stylet and does not reach its distal end. The lack of eyes also differentiates P. toryne sp. n. from P. bipartitum , P. paracochleare , and P. wehrenbergi ; however, P. balticum also lacks eyes.

The sister group of clade ‘ cochleare ’ comprises a trichotomy of P. neglectum and two monophyletic clades ( ‘ roseapharynx ’ and ‘ marmoratum ’; Fig. 22 ). Karling (1967) placed P. neglectum in a group of species characterised by a proximally coiled stylet, four of which form the clade ‘ marmoratum ’. In addition to this typical stylet, these four species also share a well-developed male copulatory bursa and a relatively small seminal receptacle, incorporated in, or positioned next to, the bursa. Other species showing this unique combination of traits are P. ensifer ( Uljanin, 1870) Pereyaslawzewa, 1892 ; P. gracile Ax, 1951 ; P. karlingi Ehlers, 1974 ; P. kristenseni Ax, 1993 ; and P. sartagine Ax & Ehlers, 1973 . However, Karling (1967) excluded P. gracile from this group because its stylet is curved, not coiled, and excluded P. ensifer from his discussion because of the lack of detailed morphology of the stylet. The phylogenetic analyses, however, cannot unambiguously confirm the position of P. neglectum within this group, as its position remains unresolved. According to Karling (1967), P. neglectum is different from the other species in this group because of the presence of a glandular organ opening near the aperture of the male atrium to the common atrium, as occurs in P. norvegicum and P. spiriferum Karling, 1967 . Unfortunately, these species were not included in the phylogenetic analysis and future studies will be needed to reveal if the presence of this glandular organ is a synapomorphy of these three species.

Because of its morphological resemblance with some species in clade ‘ marmoratum ’ ( P. caligulatum and P. meixneri ), it can be conjectured that also P. panamaense sp. n. belongs to this clade. Indeed, P. panamaense sp. n. also possesses a proximally coiled stylet and a copulatory bursa. However, the absence of a seminal receptacle in P. panamaense sp. n. distinguishes it from the other species in clade ‘ marmoratum ’. Other species resembling P. panamaense sp. n. are P. infundibulum , and P. minutum ; the former, however, lacks a copulatory bursa. The stylet is very long and involves several coils in P. meixneri (710–720 µm) and two to three coils in P. caligulatum (1.170 –1.350 µm) and P. infundibulum (296–307 µm long). In P. panamaense sp. n. and in P. minutum , the stylet involves a single coil (Fig. 13e). Promesostoma panamaense sp. n. can be distinguished from the latter (and the other three) species as its stylet is much shorter (~ 88 µm) compared to the 430–490 µm reported for P. minutum ( Ax, 1956) . Promesostoma panamaense sp. n. also differs from P. minutum in the morphology of the distal end of the stylet, which bears a wing and a pointed projection in the later species. In P. panamaense sp. n., the stylet end is oblique or rounded and no wing is present.

From the above, it is still unclear which morphological characters are indeed diagnostic for clade ‘ marmoratum ’ and which species can be included there. This can only be solved by incorporating more species into the molecular analyses and/or additional morphological markers.

The two species with a known morphology included in clade ‘ roseapharynx ’ ( P. dipterostylum and P. roseapharynx sp. n.) each possess a well-developed male copulatory bursa. Apart from that, it is difficult to pinpoint morphological characters diagnostic for this clade. Moreover, this feature is not unique for these two species (see discussion above). In P. roseapharynx sp. n., the prostate vesicle is oriented forward, entering the stylet, which shows a proximal> 90° bent. This is also the case in six other species of Promesostoma : P. alaskanum Ax & Armonies, 1990 ; P. dennisalleni Ax, 1995 ; P. digitosum Ax, 1995 ; P. fibulatum Ax & Armonies, 1987 ; P. nynaesiense Karling, 1957 ; and P. teshirogii Ax, 1992 . In two of these species, P. digitosum and P. fibulatum , the stylet is distally branched, distinguishing it clearly from the unbranched stylet of P. roseapharynx sp. n. Measuring ~ 177 µm in length, the stylet of P. roseapharynx sp. n. is shorter than that P. nynaesiense (363–399 µm), P. alaskanum (250 µm), and P. teshirogii (200 µm), but larger than that of P. dennisalleni (120–130 µm long). Furthermore, the stylet of P. dennisalleni shows an asymmetric thickening of its distal opening ( Ax, 1995) and it is proximally rolled up in P. alaskanum ( Ax & Armonies, 1990) , which is not the case in P. roseapharynx sp. n. In P. teshirogii , the copulatory bursa is elongated, oriented forward, and reaches the caudal part of the brain, whereas in P. roseapharynx sp. n., it is globular, located just anterior to the pharynx and is oriented sideways.

The stylet of Promesostoma baculum sp. n. is approximately 90° bent proximally and in that respect comparable with that of P. bilobatum Ax & Armonies, 1987 ; P. dipterostylum Karling, 1967 ; P. nynaesiense Karling, 1957 ; and P. teshirogii . The stylet of P. baculum sp. n. does not carry distinctive ornamentations, such as the distal spoon-shaped oval plates in P. bilobatum (see Ax & Armonies, 1987), or the proximal wing-like structures in P. dipterostylum (see Karling, 1967). The stylet of P. baculum sp. n. is 93 µm long, much shorter than that of P. nynaesiense (363–399 µm), P. dipterostylum and P. teshirogii (~ 200 µm), and P. bilobatum (~ 210 µm).

Figures

Fig. 22 Majority-rule consensus tree from the Bayesian analysis of the concatenated 18S rDNA+28S rDNA+cox1 dataset of Promesostoma. Branches with support values below the thresholds in the legend of the three analyses were collapsed. Symbols above branches represent posterior probabilities (: 0.95 ≤ pp <100; : 0.85 ≤ pp <0.95;: pp <0.85). Symbols below branches